Multi-armed, monofunctional, and hydrolytically stable derivatives of poly (ethylene glycol) and related polymers for modification of surfaces and molecules

ABSTRACT

Multi-armed, monofunctional, and hydrolytically stable polymers are described having the structure  
                 
 
     wherein Z is a moiety that can be activated for attachment to biologically active molecules such as proteins and wherein P and Q represent linkage fragments that join polymer arms poly a  and poly b , respectively, to central carbon atom, C, by hydrolytically stable linkages in the absence of aromatic rings in the linkage fragments. R typically is hydrogen or methyl, but can be a linkage fragment that includes another polymer arm. A specific example is an mPEG disubstituted lysine having the structure  
                 
 
     where mPEG a  and mPEG b  have the structure CH 3 O—(CH 2 CH 2 O) n CH 2 CH 2 — wherein n may be the same or different for poly a - and poly b - and can be from 1 to about 1,150 to provide molecular weights of from about 100 to 100,000.

[0001] This application is related to and claims the benefit of thefiling date of U.S. Ser. No. 08/371,065, which was filed on Jan. 10,1995 and is entitled MULTI-ARMED, MONOFUNCTIONAL, AND HYDROLYTICALLYSTABLE DERIVATIVES OF POLY(ETHYLENE GLYCOL) AND RELATED POLYMERS FORMODIFICATION OF SURFACES AND MOLECULES.

FIELD OF THE INVENTION

[0002] This invention relates to monofunctional derivatives ofpoly(ethylene glycol) and related polymers and to methods for theirsynthesis and activation for use in modifying the characteristics ofsurfaces and molecules.

BACKGROUND OF THE INVENTION

[0003] Improved chemical and genetic methods have made many enzymes,proteins, and other peptides and polypeptides available for use as drugsor biocatalysts having specific catalytic activity. However, limitationsexist to use of these compounds.

[0004] For example, enzymes that exhibit specific biocatalytic activitysometimes are less useful than they otherwise might be because ofproblems of low stability and solubility in organic solvents. During invivo use, many proteins are cleared from circulation too rapidly. Someproteins have less water solubility than is optimal for a therapeuticagent that circulates through the bloodstream. Some proteins give riseto immunological problems when used as therapeutic agents. Immunologicalproblems have been reported from manufactured proteins even where thecompound apparently has the same basic structure as the homologousnatural product. Numerous impediments to the successful use of enzymesand proteins as drugs and biocatalysts have been encountered.

[0005] One approach to the problems that have arisen in the use ofpolypeptides as drugs or biocatalysts has been to link suitablehydrophilic or amphiphilic polymer derivatives to the polypeptide tocreate a polymer cloud surrounding the polypeptide. If the polymerderivative is soluble and stable in organic solvents, then enzymeconjugates with the polymer may acquire that solubility and stability.Biocatalysis can be extended to organic media with enzyme and polymercombinations that are soluble and stable in organic solvents.

[0006] For in vivo use, the polymer cloud can help to protect thecompound from chemical attack, to limit adverse side effects of thecompound when injected into the body, and to increase the size of thecompound, potentially to render useful compounds that have somemedicinal benefit, but otherwise are not useful or are even harmful toan organism. For example, the polymer cloud surrounding a protein canreduce the rate of renal excretion and immunological complications andcan increase resistance of the protein to proteolytic breakdown intosimpler, inactive substances.

[0007] However, despite the benefits of modifying polypeptides withpolymer derivatives, additional problems have arisen. These problemstypically arise in the linkage of the polymer to the polypeptide. Thelinkage may be difficult to form. Bifunctional or multifunctionalpolymer derivatives tend to cross link proteins, which can result in aloss of solubility in water, making a polymer-modified proteinunsuitable for circulating through the blood stream of a livingorganism. Other polymer derivatives form hydrolytically unstablelinkages that are quickly destroyed on injection into the blood stream.Some linking moieties are toxic. Some linkages reduce the activity ofthe protein or enzyme, thereby rendering the protein or enzyme lesseffective.

[0008] The structure of the protein or enzyme dictates the location ofreactive sites that form the loci for linkage with polymers. Proteinsare built of various sequences of alpha-amino acids, which have thegeneral structure

[0009] The alpha amino moiety (H₂N—) of one amino acid joins to thecarboxyl moiety (—COOH) of an adjacent amino acid to form amidelinkages, which can be represented as

[0010] where n can be hundreds or thousands. The terminal amino acid ofa protein molecule contains a free alpha amino moiety that is reactiveand to which a polymer can be attached. The fragment represented by Rcan contain reactive sites for protein biological activity and forattachment of polymer.

[0011] For example, in lysine, which is an amino acid forming part ofthe backbone of most proteins, a reactive amino (—NH₂) moiety is presentin the epsilon position as well as in the alpha position. The epsilon—NH₂ is free for reaction under conditions of basic pH. Much of the arthas been directed to developing polymer derivatives having activemoieties for attachment to the epsilon —NH₂ moiety of the lysinefraction of a protein. These polymer derivatives all have in common thatthe lysine amino acid fraction of the protein typically is modified bypolymer attachment, which can be a drawback where lysine is important toprotein activity.

[0012] Poly(ethylene glycol), which is commonly referred to simply as“PEG,” has been the nonpeptidic polymer most used so far for attachmentto proteins. The PEG molecule typically is linear and can be representedstructurally as

HO—(CH₂CH₂O)_(n)CH₂CH₂—OH

[0013] or, more simply, as HO—PEG—OH. As shown, the PEG molecule isdifunctional, and is sometimes referred to as “PEG diol.” The terminalportions of the PEG molecule are relatively nonreactive hydroxylmoieties, —OH, that can be activated, or converted to functionalmoieties, for attachment of the PEG to other compounds at reactive siteson the compound.

[0014] For example, the terminal moieties of PEG diol have beenfunctionalized as active carbonate ester for selective reaction withamino moieties by substitution of the relatively nonreactive hydroxylmoieties, —OH, with succinimidyl active ester moieties from N-hydroxysuccinimide. The succinimidyl ester moiety can be representedstructurally as

[0015] Difunctional PEG, functionalized as the succinimidyl carbonate,has a structure that can be represented as

[0016] Difunctional succinimidyl carbonate PEG has been reacted withfree lysine monomer to make high molecular weight polymers. Free lysinemonomer, which is also known as alpha, epsilon diaminocaproic acid, hasa structure with reactive alpha and epsilon amino moieties that can berepresented as

[0017] These high molecular weight polymers from difunctional PEG andfree lysine monomer have multiple, pendant reactive carboxyl groupsextending as branches from the polymer backbone that can be representedstructurally as

[0018] The pendant carboxyl groups typically have been used to couplenonprotein pharmaceutical agents to the polymer. Protein pharmaceuticalagents would tend to be cross linked by the multifunctional polymer withloss of protein activity.

[0019] Multiarmed PEGs having a reactive terminal moiety on each branchhave been prepared by the polymerization of ethylene oxide onto multiplehydroxyl groups of polyols including glycerol. Coupling of this type ofmulti-functional, branched PEG to a protein normally produces across-linked product with considerable loss of protein activity.

[0020] It is desirable for many applications to cap the PEG molecule onone end with an essentially nonreactive end moiety so that the PEGmolecule is monofunctional. Monofunctional PEGs are usually preferredfor protein modification to avoid cross linking and loss of activity.One hydroxyl moiety on the terminus of the PEG diol molecule typicallyis substituted with a nonreactive methyl end moiety, CH₃—. The oppositeterminus typically is converted to a reactive end moiety that can beactivated for attachment at a reactive site on a surface or a moleculesuch as a protein.

[0021] PEG molecules having a methyl end moiety are sometimes referredto as monomethoxy-poly(ethylene glycol) and are sometimes referred tosimply as “mPEG.” The mPEG polymer derivatives can be representedstructurally as

H₃C—O—(CH₂CH₂O)_(n)—CH₂CH₂—Z

[0022] where n typically equals from about 45 to 115 and —Z is afunctional moiety that is active for selective attachment to a reactivesite on a molecule or surface or is a reactive moiety that can beconverted to a functional moiety.

[0023] Typically, mPEG polymers are linear polymers of molecular weightin the range of from about 1,000 to 5,000. Higher molecular weights havealso been examined, up to a molecular weight of about 25,000, but thesemPEGs typically are not of high purity and have not normally been usefulin PEG and protein chemistry. In particular, these high molecular weightmPEGs typically contain significant percentages of PEG diol.

[0024] Proteins and other molecules typically have a limited number anddistinct type of reactive sites available for coupling, such as theepsilon —NH₂ moiety of the lysine fraction of a protein. Some of thesereactive sites may be responsible for a protein's biological activity. APEG derivative that attached to a sufficient number of such sites toimpart the desired characteristics can adversely affect the activity ofthe protein, which offsets many of the advantages otherwise to begained.

[0025] Attempts have been made to increase the polymer cloud volumesurrounding a protein molecule without further deactivating the protein.Some PEG derivatives have been developed that have a single functionalmoiety located along the polymer backbone for attachment to anothermolecule or surface, rather than at the terminus of the polymer.Although these compounds can be considered linear, they are oftenreferred to as “branched” and are distinguished from conventional,linear PEG derivatives since these molecules typically comprise a pairof mPEG- molecules that have been joined by their reactive end moietiesto another moiety, which can be represented structurally as —T—, andthat includes a reactive moiety, —Z, extending from the polymerbackbone. These compounds have a general structure that can berepresented as

[0026] These monofunctional mPEG polymer derivatives show a branchedstructure when linked to another compound. One such branched form ofmPEG with a single active binding site, —Z, has been prepared bysubstitution of two of the chloride atoms of trichloro-s-triazine withmPEG to make mPEG-disubstituted chlorotriazine. The third chloride isused to bind to protein. An mPEG disubstituted chlorotriazine and itssynthesis are disclosed in Wada, H., Imamura, l., Sako, M., Katagiri,S., Tarui, S., Nishimura, H., and Inada, Y. (1990) Antitumor enzymes:polyethylene glycol-modified asparaginase. Ann. N.Y. Acad. Sci. 613,95-108. Synthesis of mPEG disubstituted chlorotriazine is representedstructurally below.

[0027] However, mPEG-disubstituted chlorotriazine and the procedure usedto prepare it present severe limitations because coupling to protein ishighly nonselective. Several types of amino acids other than lysine areattacked and many proteins are inactivated. The intermediate is toxic.Also, the mPEG-disubstituted chlorotriazine molecule reacts with water,thus substantially precluding purification of the branched mPEGstructure by commonly used chromatographic techniques in water.

[0028] A branched mPEG with a single activation site based on couplingof mPEG to a substituted benzene ring is disclosed in European PatentApplication Publication No. 473 084 A2. However, this structure containsa benzene ring that could have toxic effects if the structure isdestroyed in a living organism.

[0029] Another branched mPEG with a single activation site has beenprepared through a complex synthesis in which an active succinate moietyis attached to the mPEG through a weak ester linkage that is susceptibleto hydrolysis. An mPEG-OH is reacted with succinic anhydride to make thesuccinate. The reactive succinate is then activated as the succinimide.The synthesis, starting with the active succinimide, includes thefollowing steps, represented structurally below.

[0030] The mPEG activated as the succinimide, mPEG succinimidylsuccinate, is reacted in the first step as shown above with norleucine.The symbol —R in the synthesis represents the n-butyl moiety ofnorleucine. The mPEG and norleucine conjugate (A) is activated as thesuccinimide in the second step by reaction with N-hydroxy succinimide.As represented in the third step, the mPEG and norleucine conjugateactivated as the succinimide (B) is coupled to the alpha and epsilonamino moieties of lysine to create an mPEG disubstituted lysine (C)having a reactive carboxyl moiety. In the fourth step, the mPEGdisubstituted lysine is activated as the succinimide.

[0031] The ester linkage formed from the reaction of the mPEG-OH andsuccinic anhydride molecules is a weak linkage that is hydrolyticallyunstable. In vivo application is therefore limited. Also, purificationof the branched mPEG is precluded by commonly used chromatographictechniques in water, which normally would destroy the molecule.

[0032] The molecule also has relatively large molecular fragmentsbetween the carboxyl group activated as the succinimide and the mPEGmoieties due to the number of steps in the synthesis and to the numberof compounds used to create the fragments. These molecular fragments aresometimes referred to as “linkers” or “spacer arms,” and have thepotential to act as antigenic sites promoting the formation ofantibodies upon injection and initiating an undesirable immunologicalresponse in a living organism.

SUMMARY OF THE INVENTION

[0033] The invention provides a branched or “multi-armed” amphiphilicpolymer derivative that is monofunctional, hydrolytically stable, can beprepared in a simple, one-step reaction, and possesses no aromaticmoieties in the linker fragments forming the linkages with the polymermoieties. The derivative can be prepared without any toxic linkage orpotentially toxic fragments. Relatively pure polymer molecules of highmolecular weight can be created. The polymer can be purified bychromatography in water. A multi-step method can be used if it isdesired to have polymer arms that differ in molecular weight. Thepolymer arms are capped with relatively nonreactive end groups. Thederivative can include a single reactive site that is located along thepolymer backbone rather than on the terminal portions of the polymermoieties. The reactive site can be activated for selective reactions.

[0034] The multi-armed polymer derivative of the invention having asingle reactive site can be used for, among other things, proteinmodification with a high retention of protein activity. Protein andenzyme activity can be preserved and in some cases is enhanced. Thesingle reactive site can be converted to a functional group for highlyselective coupling to proteins, enzymes, and surfaces. A larger, moredense polymer cloud can be created surrounding a biomolecule with fewerattachment points to the biomolecule as compared to conventional polymerderivatives having terminal functional groups. Hydrolytically weak esterlinkages can be avoided. Potentially harmful or toxic products ofhydrolysis can be avoided. Large linker fragments can be avoided so asto avoid an antigenic response in living organisms. Cross linking isavoided.

[0035] The molecules of the invention can be represented structurally aspoly_(a)-P—CR(—Q—poly_(b))—Z or:

[0036] Poly_(a) and poly_(b) represent nonpeptidic and substantiallynonreactive water soluble polymeric arms that may be the same ordifferent. C represents carbon. P and Q represent linkage fragments thatmay be the same or different and that join polymer arms poly_(a), andpoly_(b), respectively, to C by hydrolytically stable linkages in theabsence of aromatic rings in the linkage fragments. R is a moietyselected from the group consisting of H, substantially nonreactive,usually alkyl, moieties, and linkage fragments attached by ahydrolytically stable linkage in the absence of aromatic rings to anonpeptidic and substantially nonreactive water soluble polymeric arm.The moiety —Z comprises a moiety selected from the group consisting ofmoieties having a single site reactive toward nucleophilic moieties,sites that can be converted to sites reactive toward nucleophilicmoieties, and the reaction product of a nucleophilic moiety and moietieshaving a single site reactive toward nucleophilic moieties.

[0037] Typically, the moiety —P—CR(—Q—)—Z is the reaction product of alinker moiety and the reactive site of monofunctional, nonpeptidicpolymer derivatives, poly_(a)-W and poly_(b)-W, in which W is thereactive site. Polymer arms poly_(a) and poly_(b) are nonpeptidicpolymers and can be selected from polymers that have a single reactivemoiety that can be activated for hydrolytically stable coupling to asuitable linker moiety. The linker has the general structure X—CR—(Y)—Z,in which X and Y represent fragments that contain reactive sites forcoupling to the polymer reactive site W to form linkage fragments P andQ, respectively.

[0038] In one embodiment, at least one of the polymer arms is apoly(ethylene glycol) moiety capped with an essentially nonreactive endgroup, such as a monomethoxy-poly(ethylene glycol) moiety (“mPEG-”),which is capped with a methyl end group, CH₃—. The other branch can alsobe an mPEG moiety of the same or different molecular weight, anotherpoly(ethylene glycol) moiety that is capped with an essentiallynonreactive end group other than methyl, or a different nonpeptidicpolymer moiety that is capped with a nonreactive end group such as acapped poly(alkylene oxide), a poly(oxyethylated polyol), apoly(olefinic alcohol), or others.

[0039] For example, in one embodiment poly_(a) and poly_(b) are eachmonomethoxy-poly(ethylene glycol) (“mPEG”) of the same or differentmolecular weight. The mPEG-disubstituted derivative has the generalstructure mPEG_(a)—P—CH(—Q—mPEG_(b))—Z. The moieties mPEG_(a)- andmPEG_(b)- have the structure CH₃—(CH₂CH₂O)_(n)CH₂CH₂— and n may be thesame or different for mPEG_(a) and mPEG_(b). Molecules having values ofn of from 1 to about 1,150 are contemplated.

[0040] The linker fragments P and Q contain hydrolytically stablelinkages that may be the same or different depending upon the functionalmoiety on the mPEG molecules and the molecular structure of the linkermoiety used to join the mPEG moieties in the method for synthesizing themulti-armed structure. The linker fragments typically are alkylfragments containing amino or thiol residues forming a linkage with theresidue of the functional moiety of the polymer. Depending on the degreeof substitution desired, linker fragments P and Q can include reactivesites for joining additional monofunctional nonpeptidic polymers to themulti-armed structure.

[0041] The moiety —R can be a hydrogen atom, H, a nonreactive fragment,or, depending on the degree of substitution desired, R can includereactive sites for joining additional monofunctional nonpeptidicpolymers to the multi-armed structure.

[0042] The moiety —Z can include a reactive moiety for which theactivated nonpeptidic polymers are not selective and that can besubsequently activated for attachment of the derivative to enzymes,other proteins, nucleotides, lipids, liposomes, other molecules, solids,particles, or surfaces. The moiety —Z can include a linkage fragment—R_(z). Depending on the degree of substitution desired, the R_(z)fragment can include reactive sites for joining additionalmonofunctional nonpeptidic polymers to the multi-armed structure.

[0043] Typically, the —Z moiety includes terminal functional moietiesfor providing linkages to reactive sites on proteins, enzymes,nucleotides, lipids, liposomes, and other materials. The moiety —Z isintended to have a broad interpretation and to include the reactivemoiety of monofunctional polymer derivatives of the invention, activatedderivatives, and conjugates of the derivatives with polypeptides andother substances. The invention includes biologically active conjugatescomprising a biomolecule, which is a biologically active molecule, suchas a protein or enzyme, linked through an activated moiety to thebranched polymer derivative of the invention. The invention includesbiomaterials comprising a solid such as a surface or particle linkedthrough an activated moiety to the polymer derivatives of the invention.

[0044] In one embodiment, the polymer moiety is an mPEG moiety and thepolymer derivative is a two-armed mPEG derivative based uponhydrolytically stable coupling of mPEG to lysine. The mPEG moieties arerepresented structurally as CH₃O(CH₂CH₂O)_(n)CH₂CH₂— wherein n may bethe same or different for poly_(a)- and poly_(b)- and can be from 1 toabout 1,150 to provide molecular weights of from about 100 to 100,000.The —R moiety is hydrogen. The —Z moiety is a reactive carboxyl moiety.The molecule is represented structurally as follows:

[0045] The reactive carboxyl moiety of hydrolytically stablemPEG-disubstituted lysine, which can also be called alpha, epsilon-mPEGlysine, provides a site for interacting with ion exchange chromatographymedia and thus provides a mechanism for purifying the product. Thesepurifiable, high molecular weight, monofunctional compounds have manyuses. For example, mPEG-disubstituted lysine, activated as succinimidylester, reacts with amino groups in enzymes under mild aqueous conditionsthat are compatible with the stability of most enzymes. ThemPEG-disubstituted lysine of the invention, activated as thesuccinimidyl ester, is represented as follows:

[0046] The invention includes methods of synthesizing the polymers ofthe invention. The methods comprise reacting an active suitable polymerhaving the structure poly-W with a linker moiety having the structureX—CR—(Y)Z to form poly_(a)-P—CR(—Q-poly_(b))—Z. The poly moiety in thestructure poly-W can be either poly_(a) or poly_(b) and is a polymerhaving a single reactive moiety W. The W moiety is an active moiety thatis linked to the polymer moiety directly or through a hydrolyticallystable linkage. The moieties X and Y in the structure X—CR—(Y)Z arereactive with W to form the linkage fragments Q and P, respectively. Ifthe moiety R includes reactive sites similar to those of X and Y, then Rcan also be modified with a poly-W, in which the poly can be the same asor different from poly_(a) or poly_(b). The moiety Z normally does notinclude a site that is reactive with W. However, X, Y, R, and Z can eachinclude one or more such reactive sites for preparing monofunctionalpolymer derivatives having more than two branches.

[0047] The method of the invention typically can be accomplished in oneor two steps. The method can include additional steps for preparing thecompound poly-W and for converting a reactive Z moiety to a functionalgroup for highly selective reactions.

[0048] The active Z moiety includes a reactive moiety that is notreactive with W and can be activated subsequent to formation ofpoly_(a)-P—CR(—Q-poly_(b))—Z for highly selective coupling to selectedreactive moieties of enzymes and other proteins or surfaces or anymolecule having a reactive nucleophilic moiety for which it is desiredto modify the characteristics of the molecule.

[0049] In additional embodiments, the invention provides a multi-armedmPEG derivative for which preparation is simple and straightforward.Intermediates are water stable and thus can be carefully purified bystandard aqueous chromatographic techniques. Chlorotriazine activatedgroups are avoided and more highly selective functional groups are usedfor enhanced selectivity of attachment and much less loss of activityupon coupling of the mPEG derivatives of the invention to proteins,enzymes, and other peptides. Large spacer arms between the coupledpolymer and protein are avoided to avoid introducing possible antigenicsites. Toxic groups, including triazine, are avoided. The polymerbackbone contains no hydrolytically weak ester linkages that could breakdown during in vivo applications. Monofunctional polymers of double themolecular weight as compared to the individual mPEG moieties can beprovided, with mPEG dimer structures having molecular weights of up toat least about 50,000, thus avoiding the common problem of difunctionalimpurities present in conventional, linear mPEGs.

BRIEF DESCRIPTION OF THE DRAWINGS

[0050] FIGS. 1(a), 1(b), and 1(c) illustrate the time course ofdigestion of ribonuclease (), conventional, linear mPEG-modifiedribonuclease (◯), and ribonuclease modified with a multi-armed mPEG ofthe invention (▪) as assessed by enzyme activity upon incubation withpronase (FIG. 1(a)), elastase (FIG. 1(b)), and subtilisin (FIG. 1(c)).

[0051] FIGS. 2(a) and 2(b) illustrate stability toward heat (FIG. 2(a))and pH (FIG. 2(b)) of ribonuclease (), linear mPEG-modifiedribonuclease (◯), and ribonuclease modified with a multi-armed mPEG ofthe invention (□). FIG. 2(a) is based on data taken after a 15 minuteincubation period at the indicated temperatures. FIG. 2(b) is based ondata taken over a 20 hour period at different pH values.

[0052] FIGS. 3(a) and 3(b) illustrate the time course of digestion forcatalase (); linear mPEG-modified catalase (◯), and catalase modifiedwith a multi-armed mPEG of the invention (▪) as assessed by enzymeactivity upon incubation with pronase (FIG. 3(a)) and trypsin (FIG.3(b)).

[0053]FIG. 4 illustrates the stability of catalase (), linearmPEG-modified catalase (□), and catalase modified with a multi-armedmPEG of the invention (◯) for 20 hours incubation at the indicated pHvalues.

[0054]FIG. 5 illustrates the time course of digestion of asparaginase(), linear mPEG-modified asparaginase (◯), and asparaginase modifiedwith a multi-armed mPEG of the invention (▪) as assessed by enzymeactivity assay upon trypsin incubation.

[0055]FIG. 6 illustrates the time course of autolysis of trypsin (),linear mPEG-modified trypsin (▪), and trypsin modified with amulti-armed mPEG of the invention (▴) evaluated as residual activitytowards TAME (alpha N-p-tosyl-arginine methyl ester).

DETAILED DESCRIPTION

[0056] I. Preparation of a Hydrolytically Stable mPEG-DisubstitutedLysine.

[0057] Two procedures are described for the preparation of ahydrolytically stable, two-armed, mPEG-disubstituted lysine. The firstprocedure is a two step procedure, meaning that the lysine issubstituted with each of the two mPEG moieties in separate reactionsteps. Monomethoxy-poly(ethylene glycol) arms of different lengths or ofthe same length can be substituted onto the lysine molecule, if desired,using the two step procedure. The second procedure is a one stepprocedure in which the lysine molecule is substituted with each of thetwo mPEG moieties in a single reaction step. The one step procedure issuitable for preparing mPEG-disubstituted lysine having mPEG moieties ofthe same length.

[0058] Unlike prior multisubstituted structures, no aromatic ring ispresent in the linkage joining the nonpeptidic polymer arms produced byeither the one or two step methods described below that could result intoxicity if the molecule breaks down in vivo. No hydrolytically weakester linkages are present in the linkage. Lengthy linkage chains thatcould promote an antigenic response are avoided.

[0059] The terms “group,” “functional group,” “moiety,” “active moiety,”“reactive site,” “radical,” and similar terms are somewhat synonymous inthe chemical arts and are used in the art and herein to refer todistinct, definable portions or units of a molecule or fragment of amolecule. “Reactive site,” “functional group,” and “active moiety” referto units that perform some function or have a chemical activity and arereactive with other molecules or portions of molecules. In this sense aprotein or a protein residue can be considered as a molecule and as afunctional moiety when coupled to a polymer. A polymer, such asmPEG—COOH has a reactive site, the carboxyl moiety, —COOH, that can beconverted to a functional group for selective reactions and attachmentto proteins and linker moieties. The converted polymer is said to beactivated and to have an active moiety, while the —COOH group isrelatively nonreactive in comparison to an active moiety.

[0060] The term “nonreactive” is used herein primarily to refer to amoiety that does not readily react chemically with other moieties, suchas the methyl alkyl moiety. However, the term “nonreactive” should beunderstood to exclude carboxyl and hydroxyl moieties, which, althoughrelatively nonreactive, can be converted to functional groups that areof selective reactivity.

[0061] The term “biologically active” means a substance, such as aprotein, lipid, or nucleotide that has some activity or function in aliving organism or in a substance taken from a living organism. Forexample, an enzyme can catalyze chemical reactions. The term“biomaterial” is somewhat imprecise, and is used herein to refer to asolid material or particle or surface that is compatible with livingorganisms or tissue or fluids. For example, surfaces that contact blood,whether in vitro or in vivo, can be made nonfouling by attachment of thepolymer derivatives of the invention so that proteins do not becomeattached to the surface.

[0062] A. Two Step Procedure

[0063] For the two step procedure, an activated mPEG is prepared forcoupling to free lysine monomer and then the lysine monomer isdisubstituted with the activated mPEG in two steps. The first stepoccurs in aqueous buffer. The second step occurs in dry methylenechloride. The active moiety of the mPEG for coupling to the lysinemonomer can be selected from a number of activating moieties havingleaving moieties that are reactive with the amino moieties of lysinemonomer. A commercially available activated mPEG,mPEG-p-nitrophenylcarbonate, the preparation of which is discussedbelow, was used to exemplify the two step procedure.

[0064] The two step procedure can be represented structurally asfollows:

[0065] Step 1. Preparation of mPEG-monosubstituted lysine. Modificationof a single lysine amino group was accomplished withmPEG-p-nitrophenylcarbonate in aqueous solution where both lysine andmPEG-p-nitrophenylcarbonate are soluble. The mPEG-p-nitrophenylcarbonatehas only limited stability in aqueous solution. However, lysine is notsoluble in organic solvents in which the activated mPEG is stable.Consequently, only one lysine amino group is modified by this procedure.NMR confirms that the epsilon amino group is modified. Nevertheless, theprocedure allows ready chloroform extraction of mPEG-monosubstitutedlysine from unreacted lysine and other water soluble by-products, and sothe procedure provides a desirable monosubstituted product fordisubstitution.

[0066] To prepare the mPEG-monosubstituted lysine, 353 milligrams oflysine, which is about 2.5 millimoles, was dissolved in 20 millilitersof water at a pH of about 8.0 to 8.3. Five grams ofmPEG-p-nitrophenylcarbonate of molecular weight 5,000, which is about 1millimole, was added in portions over 3 hours. The pH was maintained at8.3 with 0.2 N NaOH. The reaction mixture was stirred overnight at roomtemperature. Thereafter, the reaction mixture was cooled to 0° C. andbrought to a pH of about 3 with 2 N HCl. Impurities were extracted withdiethyl ether. The mPEG monosubstituted lysine, having the mPEGsubstituted at the epsilon amino group of lysine as confirmed by NMRanalysis, was extracted three times with chloroform. The solution wasdried. After concentration, the solution was added drop by drop todiethyl ether to form a precipitate. The precipitate was collected andthen crystallized from absolute ethanol. The percentage of modifiedamino groups was 53%, calculated by calorimetric analysis.

[0067] Step 2. Preparation of mPEG-Disubstituted Lysine. ThemPEG-monosubstituted lysine product from step 1 above is soluble inorganic solvents and so modification of the second lysine amino moietycan be achieved by reaction in dry methylene chloride. Activated mPEG,mPEG-p-nitrophenylcarbonate, is soluble and stable in organic solventsand can be used to modify the second lysine amino moiety.

[0068] Triethylamine (“TEA”) was added to 4.5 grams ofmPEG-monosubstituted lysine, which is about 0.86 millimoles. The mixtureof TEA and mPEG-monosubstituted lysine was dissolved in 10 millilitersof anhydrous methylene chloride to reach a pH of 8.0. Four and ninetenths grams of mPEG-p-nitrophenycarbonate of molecular weight 5,000,which is 1.056 millimoles, was added over 3 hours to the solution. If itis desirable to make an mPEG disubstituted compound having mPEG arms ofdifferent lengths, then a different molecular weight mPEG could havebeen used. The pH was maintained at 8.0 with TEA. The reaction mixturewas refluxed for 72 hours, brought to room temperature, concentrated,filtered, precipitated with diethyl ether and then crystallized in aminimum amount of hot ethanol. The excess of activated mPEG,mPEG-p-nitrophenycarbonate, was deactivated by hydrolysis in an alkalineaqueous medium by stirring overnight at room temperature. The solutionwas cooled to 0° C. and brought to a pH of about 3 with 2 N HCl.

[0069] p-Nitrophenol was removed by extraction with diethyl ether.Monomethyl-poly(ethylene glycol)-disubstituted lysine and remainingtraces of mPEG were extracted from the mixture three times withchloroform, dried, concentrated, precipitated with diethyl ether andcrystallized from ethanol. No unreacted lysine amino groups remained inthe polymer mixture as assessed by calorimetric analysis.

[0070] Purification of mPEG-disubstituted lysine and removal of mPEGwere accomplished by gel filtration chromatography using a Bio Gel P100(Bio-Rad) column. The column measured 5 centimeters by 50 centimeters.The eluent was water. Fractions of 10 milliliters were collected. Up to200 milligrams of material could be purified for each run. The fractionscorresponding to mPEG-disubstituted lysine were revealed by iodinereaction. These fractions were pooled, concentrated, and then dissolvedin ethanol and concentrated. The mPEG-disubstituted lysine product wasdissolved in methylene chloride, precipitated with diethyl ether, andcrystallized from ethanol.

[0071] The mPEG-disubstituted lysine was also separated from unmodifiedmPEG-OH and purified by an alternative method. Ion exchangechromatography was performed on a QAE Sephadex ASO column (Pharmacia)that measured 5 centimeters by 80 centimeters. An 8.3 mM borate bufferof pH 8.9 was used. This alternative procedure permitted fractionationof a greater amount of material per run than the other method abovedescribed (up to four grams for each run).

[0072] For both methods of purification, purified mPEG-disubstitutedlysine of molecular weight 10,000, titrated with NaOH, showed that 100%of the carboxyl groups were free carboxyl groups. These results indicatethat the reaction was complete and the product pure.

[0073] The purified mPEG-disubstituted lysine was also characterized by¹H—NMR on a 200 MHz Bruker instrument in dimethyl sulfoxide, d6, at a 5%weight to volume concentration. The data confirmed the expectedmolecular weight of 10,000 for the polymer. The chemical shifts andassignments of the protons in the mPEG-disubstituted lysine are asfollows: 1.2-1.4 ppm (multiplet, 6H, methylenes 3,4,5 of lysine); 1.6ppm (multiplet, 2H, methylene 6 of lysine); 3.14 ppm (s, 3H, terminalmPEG methoxy); 3.49 ppm (s, mPEG backbone methylene); 4.05 ppm (t, 2H,—CH₂, —OCO—); 7.18 ppm (t, 1H, —NH— lysine); and 7.49 ppm (d,1H, —NH—lysine).

[0074] The above signals are consistent with the reported structuresince two different carbamate NH protons are present. The firstcarbamate NH proton (at 7.18 ppm) shows a triplet for coupling with theadjacent methylene group. The second carbamate NH proton (at 7.49 ppm)shows a doublet because of coupling with the α-CH of lysine. Theintensity of these signals relative to the mPEG methylene peak isconsistent with the 1:1 ratio between the two amide groups and theexpected molecular weight of 10,000 for the polymer.

[0075] The two step procedure described above allows polymers ofdifferent types and different lengths to be linked with a singlereactive site between them. The polymer can be designed to provide apolymer cloud of custom shape for a particular application.

[0076] The commercially available activated mPEG,mPEG-p-nitrophenylcarbonate, is available from Shearwater Polymers, Inc.in Huntsville, Ala. This compound was prepared by the followingprocedure, which can be represented structurally as follows:

[0077] Five grams of mPEG-OH of molecular weight 5,000, or 1 millimole,were dissolved in 120 milliliters of toluene and dried azeotropicallyfor 3 hours. The solution was cooled to room temperature andconcentrated under vacuum. Reactants added to the concentrated solutionunder stirring at 0° C. were 20 milliliters of anhydrous methylenechloride and 0.4 g of p-nitrophenylchloroformate, which is 2 millimoles.The pH of the reaction mixture was maintained at 8 by adding 0.28milliliters of triethylamine (“TEA”), which is 2 millimoles. Thereaction mixture was allowed to stand overnight at room temperature.Thereafter, the reaction mixture was concentrated under vacuum to about10 milliliters, filtered, and dropped into 100 milliliters of stirreddiethyl ether. A precipitate was collected from the diethyl ether byfiltration and crystallized twice from ethyl acetate. Activation of mPEGwas determined to be 98%. Activation was calculatedspectrophotometrically on the basis of the absorption at 400 nm inalkaline media after 15 minutes of released 4-nitrophenol (ε ofp-nitrophenol at 400 nm equals 17,000).

[0078] B. One Step Procedure

[0079] In the one step procedure, mPEG disubstituted lysine is preparedfrom lysine and an activated mPEG in a single step as representedstructurally below:

[0080] Except for molecular weight attributable to a longer PEG backbonein the activated mPEG used in the steps below, the mPEG disubstitutedlysine of the one step procedure does not differ structurally from themPEG disubstituted lysine of the two step procedure. It should berecognized that the identical compound, having the same molecularweight, can be prepared by either method.

[0081] Preparation of mPEG disubstituted lysine by the one stepprocedure proceeded as follows: Succinimidylcarbonate mPEG of molecularweight about 20,000 was added in an amount of 10.8 grams, which is5.4×10⁻⁴ moles, to 40 milliliters of lysine HCl solution. The lysine HCLsolution was in a borate buffer of pH 8.0. The concentration was 0.826milligrams succinimidylcarbonate mPEG per milliliter of lysine HCLsolution, which is 1.76×10⁻⁴ moles. Twenty milliliters of the samebuffer was added. The solution pH was maintained at 8.0 with aqueousNaOH solution for the following 8 hours. The reaction mixture wasstirred at room temperature for 24 hours.

[0082] Thereafter, the solution was diluted with 300 milliliters ofdeionized water. The pH of the solution was adjusted to 3.0 by theaddition of oxalic acid. The solution was then extracted three timeswith dichloromethane. The combined dichloromethane extracts were driedwith anhydrous sodium sulphate and filtered. The filtrate wasconcentrated to about 30 milliliters. The product, an impure mPEGdisubstituted lysine, was precipitated with about 200 milliliters ofcold ethyl ether. The yield was 90%.

[0083] Nine grams of the above impure mPEG-disubstituted lysine reactionproduct was dissolved in 4 liters of distilled water and then loadedonto a column of DEAE Sepharose FF, which is 500 milliliters of gelequilibrated with 1500 milliliters of boric acid in a 0.5% sodiumhydroxide buffer at a pH of 7.0. The loaded system was then washed withwater. Impurities of succinimidylcarbonate mPEG and mPEG-monosubstitutedlysine, both of molecular weight about 20,000, were washed off thecolumn. However, the desired mPEG disubstituted lysine of molecularweight 20,000 was eluted with 10 mM NaCl. The pH of the eluate wasadjusted to 3.0 with oxalic acid and then mPEG disubstituted lysine wasextracted with dichloromethane, dried with sodium sulphate,concentrated, and precipitated with ethyl ether. Five and one tenthgrams of purified mPEG disubstituted lysine were obtained. The molecularweight was determined to be 38,000 by gel filtration chromatography and36,700 by potentiometric titration.

[0084] The one step procedure is simple in application and is useful forproducing high molecular weight dimers that have polymers of the sametype and length linked with a single reactive site between them.

[0085] Additional steps are represented below for preparingsuccinimidylcarbonate mPEG for disubstitution of lysine.

[0086] Succinimidylcarbonate mPEG was prepared by dissolving 30 grams ofmPEG-OH of molecular weight 20,000, which is about 1.5 millimoles, in120 milliliters of toluene. The solution was dried azeotropically for 3hours. The dried solution was cooled to room temperature. Added to thecooled and dried solution were 20 milliliters of anhydrousdichloromethane and 2.33 milliliters of a 20% solution of phosgene intoluene. The solution was stirred continuously for a minimum of 16 hoursunder a hood due to the highly toxic fumes.

[0087] After distillation of excess phosgene and solvent, the remainingsyrup, which contained mPEG chlorocarbonate, was dissolved in 100milliliters of anhydrous dichloromethane, as represented above. To thissolution was added 3 millimoles of triethylamine and 3 millimoles ofN-hydroxysuccinimide. The reaction mixture remained standing at roomtemperature for 24 hours. Thereafter, the solution was filtered througha silica gel bed of pore size 60 Angstroms that had been wetted withdichloromethane. The filtrate was concentrated to 70 milliliters.Succinimidylcarbonate mPEG of molecular weight about 20,000 wasprecipitated in ethyl ether and dried in vacuum for a minimum of 8hours. The yield was 90%. Succinimidylcarbonate-mPEG is availablecommercially from Shearwater Polymers in Huntsville, Ala.

[0088] The mPEG disubstituted lysine of the invention can be representedstructurally more generally as poly_(a)-P—CR (—Q-poly_(b))—Z or:

[0089] For the mPEG disubstituted lysines described above, —P—CR(—Q—)—Zis the reaction product of a precursor linker moiety having two reactiveamino groups and active monofunctional precursors of poly_(a) andpoly_(b) that have been joined to the linker moiety at the reactiveamino sites. Linker fragments Q and P contain carbamate linkages formedby joining the amino containing portions of the lysine molecule with thefunctional group with which the mPEG was substituted. The linkerfragments are selected from —O—C(O)NH(CH₂)₄— and —O—C(O)NH— and aredifferent in the exemplified polymer derivative. However, it should berecognized that P and Q could both be —O—C(O)NH(CH₂)₄— or —O—C(O)NH— orsome other linkage fragment, as discussed below. The moiety representedby R is hydrogen, H. The moiety represented by Z is the carboxyl group,— COOH. The moieties P, R, Q, and Z are all joined to a central carbonatom.

[0090] The nonpeptidic polymer arms, poly_(a) and poly_(b), are mPEGmoieties mPEG_(a) and mPEG_(b), respectively, and are the same on eachof the linker fragments Q and P for the examples above. The mPEGmoieties have a structure represented as CH₃O—(CH₂CH₂O)_(n)CH₂CH₂—. Forthe mPEG disubstituted lysine made by the one step method, n is about454 to provide a molecular weight for each mPEG moiety of 20,000 and adimer molecular weight of 40,000. For the mPEG disubstituted lysine madeby the two step method, n is about 114 to provide a molecular weight foreach mPEG moiety of 5,000 and a dimer molecular weight of 10,000.

[0091] Lysine disubstituted with mPEG and having as dimer molecularweights of 10,000 and 40,000 and procedures for preparation ofmPEG-disubstituted lysine have been shown. However, it should berecognized that mPEG disubstituted lysine and other multi-armedcompounds of the invention can be made in a variety of molecularweights, including ultra high molecular weights. High molecular weightmonofunctional PEGs are otherwise difficult to obtain.

[0092] Polymerization of ethylene oxide to yield mPEGs usually producesmolecular weights of up to about 20,000 to 25,000 g/mol. Accordingly,two-armed mPEG disubstituted lysines of molecular weight of about 40,000to 50,000 can be made according to the invention. Higher molecularweight lysine disubstituted PEGs can be made if the chain length of thelinear mPEGs is increased, up to about 100,000. Higher molecular weightscan also be obtained by adding additional monofunctional nonpeptidicpolymer arms to additional reactive sites on a linker moiety, withinpractical limits of steric hindrance. However, no unreacted active siteson the linker should remain that could interfere with themonofunctionality of the multi-armed derivative. Lower molecular weightdisubstituted mPEGs can also be made, if desired, down to a molecularweight of about 100 to 200.

[0093] It should be recognized that a wide variety of linker fragments Pand Q are available, although not necessarily with equivalent results,depending on the precursor linker moiety and the functional moiety withwhich the activated mPEG or other nonpeptidic monofunctional polymer issubstituted and from which the linker fragments result. Typically, thelinker fragments will contain the reaction products of portions oflinker moieties that have reactive amino and/or thiol moieties andsuitably activated nonpeptidic, monofunctional, water soluble polymers.

[0094] For example, a wide variety of activated mPEGs are available thatform a wide variety of hydrolytically stable linkages with reactiveamino moieties. Linkages can be selected from the group consisting ofamide, amine, ether, carbamate, which are also called urethane linkages,urea, thiourea, thiocarbamate, thiocarbonate, thioether, thioester,dithiocarbonate linkages, and others. However, hydrolytically weak esterlinkages and potentially toxic aromatic moieties are to be avoided.

[0095] Hydrolytic stability of the linkages means that the linkagesbetween the polymer arms and the linker moiety are stable in water andthat the linkages do not react with water at useful pHs for an extendedperiod of time of at least several days, and potentially indefinitely.Most proteins could be expected to lose their activity at a caustic pHof 11 or higher, so the derivatives should be stable at a pH of lessthan about 11.

[0096] Examples of the above linkages and their formation from activatedmPEG and lysine are represented structurally below.

[0097] One or both of the reactive amino moieties, —NH₂, of lysine oranother linker moiety can be replaced with thiol moieties, —SH. Wherethe linker moiety has a reactive thiol moiety instead of an aminomoiety, then the linkages can be selected from the group consisting ofthioester, thiocarbonate, thiocarbamate, dithiocarbamate, thioetherlinkages, and others. The above linkages and their formation fromactivated mPEG and lysine in which both amino moieties have beenreplaced with thiol moieties are represented structurally below.

[0098] It should be apparent that the mPEG or other monofunctionalpolymer reactants can be prepared with a reactive amino moiety and thenlinked to a suitable linker moiety having reactive groups such as thoseshown above on the mPEG molecule to form hydrolytically stable linkagesas discussed above. For example, the amine linkage could be formed asfollows:

[0099] Examples of various active electrophilic moieties useful foractivating polymers or linking moieties for biological and biotechnicalapplications in which the active moiety is reacted to formhydrolytically stable linkages in the absence of aromatic moietiesinclude trifluoroethylsulfonate, isocyanate, isosthiocyanate, activeesters, active carbonates, various aldehydes, various sulfones,including chloroethylsulfone and vinylsulfone, maleimide, iodoacetamide,and iminoesters. Active esters include N-hydroxylsuccinimidyl ester.Active carbonates include N-hydroxylsuccinimidyl carbonate,p-nitrophenylcarbonate, and trichlorophenylcarbonate. Theseelectrophilic moieties are examples of those that are suitable as Ws inthe structure poly-W and as Xs and Ys in the linker structureX—CR(—Y)—Z.

[0100] Nucleophilic moieties for forming the linkages can be amino,thiol, and hydroxyl. Hydroxyl moieties form hydrolytically stablelinkages with isocyanate electrophilic moieties. Also, it should berecognized that the linker can be substituted with differentnucleophilic or electrophilic moieties or both electrophilic andnucleophilic moieties depending on the active moieties on themonofunctional polymers with which the linker moiety is to besubstituted.

[0101] Linker moieties other than lysine are available for activationand for disubstitution or multisubstitution with mPEG and relatedpolymers for creating multi-armed structures in the absence of aromaticmoieties in the structure and that are hydrolytically stable. Examplesof such linker moieties include those having more than one reactive sitefor attachment of various monofunctional polymers.

[0102] Linker moieties can be synthesized to include multiple reactivesites such as amino, thiol, or hydroxyl groups for joining multiplesuitably activated mPEGs or other nonpeptidic polymers to the moleculeby hydrolytically stable linkages, if it is desired to design a moleculehaving multiple nonpeptidic polymer branches extending from one or moreof the linker arm fragments. The linker moieties should also include areactive site, such as a carboxyl or alcohol moiety, represented as —Zin the general structure above, for which the activated polymers are notselective and that can be subsequently activated for selective reactionsfor joining to enzymes, other proteins, surfaces, and the like.

[0103] For example, one suitable linker moiety is a diamino alcoholhaving the structure

[0104] The diamino alcohol can be disubstituted with activated mPEG orother suitable activated polymers similar to disubstitution of lysineand then the hydroxyl moiety can be activated as follows:

[0105] Other diamino alcohols and alcohols having more than two amino orother reactive groups for polymer attachment are useful. A suitablyactivated mPEG or other monofunctional, nonpeptidic, water solublepolymer can be attached to the amino groups on such a diamino alcoholsimilar to the method by which the same polymers are attached to lysineas shown above. Similarly, the amino groups can be replaced with thiolor other active groups as discussed above. However, only one hydroxylgroup, which is relatively nonreactive, should be present in the —Zmoiety, and can be activated subsequent to polymer substitution.

[0106] The moiety —Z can include a reactive moiety or functional group,which normally is a carboxyl moiety, hydroxyl moiety, or activatedcarboxyl or hydroxyl moiety. The carboxyl and hydroxyl moieties aresomewhat nonreactive as compared to the thiol, amino, and other moietiesdiscussed above. The carboxyl and hydroxyl moieties typically remainintact when the polymer arms are attached to the linker moiety and canbe subsequently activated. The carboxyl and hydroxyl moieties alsoprovide a mechanism for purification of the multisubstituted linkermoiety. The carboxyl and hydroxyl moieties provide a site forinteracting with ion exchange chromatography media.

[0107] The moiety —Z may also include a linkage fragment, represented asR_(z) in the moiety, which can be substituted or unsubstituted, branchedor linear, and joins the reactive moiety to the central carbon. Where areactive group of the —Z moiety is carboxyl, for activation aftersubstitution with nonpeptidic polymers, then the —Z moiety has thestructure, —R_(z)—COOH if the R_(z) fragment is present. For hydroxyl,the structure is —R_(z)—OH. For example, in the diamino alcoholstructure discussed above, R_(z) is CH₂. It should be understood thatthe carboxyl and hydroxyl moieties normally will extend from the R_(z)terminus, but need not necessarily do so.

[0108] R_(z) can also include the reaction product of one or morereactive moieties including reactive amino, thiol, or other moieties,and a suitably activated mPEG arm or related nonpeptidic polymer arm. Inthe latter event, R_(z) can have the structure (—L-poly_(c))—COOH or(—L-poly_(c))—OH in which —L— is the reaction product of a portion ofthe linker moiety and a suitably activated nonpeptidic polymer,poly_(c)-W, which is selected from the same group as poly_(a)-W andpoly_(b)-W but can be the same or different from poly_(a)-W andpoly_(b)-W.

[0109] It is intended that —Z have a broad definition. The moiety —Z isintended to represent not only the reactive site of the multisubstitutedpolymeric derivative that subsequently can be converted to an activeform and its attachment to the central carbon, but the activatedreactive site and also the conjugation of the precursor activated sitewith another molecule, whether that molecule be an enzyme, other proteinor polypeptide, a phospholipid, a preformed liposome, or on a surface towhich the polymer derivative is attached.

[0110] The skilled artisan should recognize that Z encompasses thecurrently known activating moieties in PEG chemistry and theirconjugates. It should also be recognized that, although the linkerfragments represented by Q and P and R_(z) should not contain aromaticrings or hydrolytically weak linkages such as ester linkages, such ringsand such hydrolytically weak linkages may be present in the active sitemoiety of —Z or in a molecule joined to such active site. It may bedesirable in some instances to provide a linkage between, for example, aprotein or enzyme and a multisubstituted polymer derivative that haslimited stability in water. Some amino acids contain aromatic moieties,and it is intended that the structure Z include conjugates ofmultisubstituted monofunctional polymer derivatives with such moleculesor portions of molecules. Activated Zs and Zs including attachedproteins and other moieties are discussed below.

[0111] When lysine, the diamino alcohol shown above, or many othercompounds are linkers, then the central carbon has a nonreactivehydrogen, H, attached thereto. In the general structurepoly_(a)-P—CR(—Q-poly_(b))—Z, R is H. It should be recognized that themoiety R can be designed to have another substantially nonreactivemoiety, such as a nonreactive methyl or other alkyl group, or can be thereaction product of one or more reactive moieties including reactiveamino, thiol, or other moieties, and a suitably activated mPEG arm orrelated nonpeptidic polymer arm. In the latter event, R can have thestructure —M-poly_(d), in which —M— is the reaction product of a portionof the linker moiety and a suitably activated nonpeptidic polymer,poly_(d)-W, which is selected from the same group as poly_(a)-W andpoly_(b)-W but can be the same or different from poly_(a)-W andpoly_(b)-W.

[0112] For example, multi-armed structures can be made having one ormore mPEGs or other nonpeptidic polymer arms extending from each portionP, Q, R, and R_(z), all of which portions extend from a central carbonatom, C, which multi-armed structures have a single reactive site forsubsequent activation included in the structure represented by Z. Uponat least the linker fragments P and Q are located at least one activesite for which the monofunctional, nonpeptidic polymers are selective.These active sites include amino moieties, thiol moieties, and othermoieties as described above.

[0113] The nonpeptidic polymer arms tend to mask antigenic properties ofthe linker fragment, if any. A linker fragment length of from 1 to 10carbon atoms or the equivalent has been determined to be useful to avoida length that could provide an antigenic site. Also, for all the linkerfragments P, Q, R, and R_(z), there should be an absence of aromaticmoieties in the structure and the linkages should be hydrolyticallystable.

[0114] Poly(ethylene glycol) is useful in the practice of the inventionfor the nonpeptidic polymer arms attached to the linker fragments. PEGis used in biological applications because it has properties that arehighly desirable and is generally approved for biological orbiotechnical applications. PEG typically is clear, colorless, odorless,soluble in water, stable to heat, inert to many chemical agents, doesnot hydrolyze or deteriorate, and is nontoxic. Poly(ethylene glycol) isconsidered to be biocompatible, which is to say that PEG is capable ofcoexistence with living tissues or organisms without causing harm. Morespecifically, PEG is not immunogenic, which is to say that PEG does nottend to produce an immune response in the body. When attached to amoiety having some desirable function in the body, the PEG tends to maskthe moiety and can reduce or eliminate any immune response so that anorganism can tolerate the presence of the moiety. Accordingly, theactivated PEGs of the invention should be substantially non-toxic andshould not tend substantially to produce an immune response or causeclotting or other undesirable effects.

[0115] The term “PEG” is used in the art and herein to describe any ofseveral condensation polymers of ethylene glycol having the generalformula represented by the structure

HO—(CH₂CH₂O)_(n)CH₂CH₂—OH

[0116] or, more simply, as HO—PEG—OH. PEG is also known aspolyoxyethylene, polyethylene oxide, polyglycol, and polyether glycol.PEG can be prepared as copolymers of ethylene oxide and many othermonomers.

[0117] Other water soluble polymers than PEG are suitable for similarmodification to create multi-armed structures that can be activated forselective reactions. These other polymers include poly(vinyl alcohol)(“PVA”); other poly(alkylene oxides) such as poly(propylene glycol)(“PPG”) and the like; and poly(oxyethylated polyols) such aspoly(oxyethylated glycerol), poly(oxyethylated sorbitol), andpoly(oxyethylated glucose), and the like. The polymers can behomopolymers or random or block copolymers and terpolymers based on themonomers of the above polymers, straight chain or branched, orsubstituted or unsubstituted similar to mPEG and other capped,monofunctional PEGs having a single active site available for attachmentto a linker.

[0118] Specific examples of suitable additional polymers includepoly(oxazoline), poly(acryloylmorpholine) (“PAcM”), andpoly(vinylpyrrolidone)(“PVP”). PVP and poly(oxazoline) are well knownpolymers in the art and their preparation and use in the synthesesdescribed above for mPEG should be readily apparent to the skilledartisan.

[0119] An example of the synthesis of a PVP disubstituted lysine havinga single carboxyl moiety available for activation is shown below. Thedisubstituted compound can be purified, activated, and used in variousreactions for modification of molecules and surfaces similarly to themPEG-disubstituted lysine described above.

[0120] Poly(acryloylmorpholine) “(PAcM)” functionalized at one end is anew polymer, the structure, preparation, and characteristics of whichare described in Italian Patent Application No. MI 92 A 0 0002616, whichwas published May 17, 1994 and is entitled, in English, “Polymers OfN-Acryloylmorpholine Functionalized At One End And Conjugates WithBioactive Materials And Surfaces.” Dimer polymers of molecular weight upto at least about 80,000 can be prepared using this polymer. Thecontents of the Italian patent application are incorporated herein byreference.

[0121] PAcM can be used similarly to mPEG or PVP to create multi-armedstructures and ultra-high molecular weight polymers. An example of aPAcM-disubstituted lysine having a single carboxyl moiety available foractivation is shown below. The disubstituted compound can be purified,activated, and used in various reactions for modification of moleculesand surfaces similarly to the mPEG- and PVP-disubstituted lysinesdescribed above.

[0122] It should also be recognized that the multi-armed monofunctionalpolymers of the invention can be used for attachment to a linker moietyto create a highly branched monofunctional structure, within thepractical limits of steric hindrance.

[0123] II. Activation of mPEG-Disubstituted Lysine and Modification ofProtein Amino Groups.

[0124] Schemes are represented below for activating themPEG-disubstituted lysine product made by either the one step or twostep procedures and for linking the activated mPEG-disubstituted lysinethrough a stable carbamate linkage to protein amino groups to preparepolymer and protein conjugates. Various other multisubstituted polymerderivatives as discussed above can be activated similarly.

[0125] A. Activation of mPEG Disubstituted Lysine.

[0126] Purified mPEG-disubstituted lysine produced in accordance withthe two step procedure discussed above was activated withN-hydroxysuccinimide to produce mPEG-disubstituted lysine activated asthe succinimidyl ester. The reaction is represented structurally below:

[0127] Six and two tenths grams of mPEG-disubstituted lysine ofmolecular weight 10,000, which is about 0.6 millimoles, was dissolved in10 milliliters of anhydrous methylene chloride and cooled to 0° C.N-hydroxysuccinimide and N,N-dicyclohexylcarbodiimide (“DCC”) were addedunder stirring in the amounts, respectively, of 0.138 milligrams, whichis about 1.2 millimoles, and 0.48 milligrams, which is about 1.2millimoles. The reaction mixture was stirred overnight at roomtemperature. Precipitated dicyclohexylurea was removed by filtration andthe solution was concentrated and precipitated with diethyl ether. Theproduct, mPEG disubstituted lysine activated as the succinimidyal ester,was crystallized from ethyl acetate. The yield of esterification,calculated on the basis of hydroxysuccinimide absorption at 260 nm(produced by hydrolysis), was over 97% (ε of hydroxysuccinimide at 260nm=8,000 m⁻¹cm⁻¹). The NMR spectrum was identical to that of theunactivated carboxylic acid except for the new succinimide singlet at2.80 ppm (2Hs)

[0128] The procedure previously described for the activation of themPEG-disubstituted lysine of molecular weight 10,000 was also followedfor the activation of the higher molecular weight polymer of molecularweight approximately 40,000 that was produced in accordance with the onestep procedure discussed above. The yield was over 95% of high molecularweight mPEG-disubstituted lysine activated as the succinimidyal ester.

[0129] It should be recognized that a number of activating groups can beused to activate the multisubstituted polymer derivatives for attachmentto surfaces and molecules. Any of the activating groups of the knownderivatives of PEG can be applied to the multisubstituted structure. Forexample, the mPEG-disubstituted lysine of the invention wasfunctionalized by activation as the succinimidyl ester, which can beattached to protein amino groups. However, there are a wide variety offunctional moieties available for activation of carboxilic acid polymermoieties for attachment to various surfaces and molecules. Examples ofactive moieties used for biological and biotechnical applicationsinclude trifluoroethylsulfonate, isocyanate, isosthiocyanate, activeesters, active carbonates, various aldehydes, various sulfones,including chloroethylsulfone and vinylsulfone, maleimide, iodoacetamide,and iminoesters. Active esters include N-hydroxylsuccinimidyl ester.Active carbonates include N-hydroxylsuccinimidyl carbonate,p-nitrophenylcarbonate, and trichlorophenylcarbonate.

[0130] A highly useful, new activating group that can be used for highlyselective coupling with thiol moieties instead of amino moieties onmolecules and surfaces is the vinyl sulfone moiety described inco-pending U.S. patent application Ser. No. 08/151,481, which was filedon Nov. 12, 1993, the contents of which are incorporated herein byreference. Various sulfone moieties can be used to activate amulti-armed structure in accordance with the invention for thiolselective coupling.

[0131] Various examples of activation of —Z reactive moieties to created—Z activated moieties are presented as follows:

[0132] It should also be recognized that, although the linker fragmentsrepresented by Q and P should not contain aromatic rings orhydrolytically weak linkages such as ester linkages, such rings and suchhydrolytically weak linkages may be present in the moiety represented by—Z. It may be desirable in some instances to provide a linkage between,for example, a protein or enzyme and a multisubstituted polymerderivative that has limited stability in water. Some amino acids containaromatic moieties, and it is intended that the structure —Z includeconjugates of multisubstituted monofunctional polymer derivatives withsuch molecules or portions of molecules.

[0133] B. Enzyme Modification

[0134] Enzymes were modified with activated, two-armed,mPEG-disubstituted lysine of the invention of molecular weight about10,000 that had been prepared according to the two step procedure andactivated as the succinimidyl ester as discussed above. The reaction isrepresented structurally below:

[0135] For comparison, enzymes were also modified with activated,conventional, linear mPEG of molecular weight 5,000, which was mPEG witha norleucine amino acid spacer arm activated as the succinimide. In thediscussion of enzyme modification below, conventional, linear mPEGderivatives with which enzymes are modified are referred to as “linearmPEG.” The activated, two-armed, mPEG-disubstituted lysine of theinvention is referred to as “two-armed mPEG.” Different procedures wereused for enzyme modification depending upon the type of enzyme and thepolymer used so that a similar extent of amino group modification orattachment for each enzyme could be obtained. Generally, higher molarratios of the two-armed mPEG were used. However, in all examples theenzymes were dissolved in a 0.2 M borate buffer of pH 8.5 to dissolveproteins. The polymers were added in small portions for about 10 minutesand stirred for over 1 hour. The amount of polymer used for modificationwas calculated on the basis of available amino groups in the enzyme.

[0136] Ribonuclease in a concentration of 1.5 milligrams per milliliterof buffer was modified at room temperature. Linear and two-armed mPEGsas described were added at a molar ratio of polymer to protein aminogroups of 2.5:1 and 5:1, respectively. Ribonuclease has a molecularweight of 13,700 D and 11 available amino groups. Catalase has amolecular weight of 250,000 D with 112 available amino groups. Trypsinhas a molecular weight of 23,000 D with 16 available amino groups.Erwinia Caratimora asparaginase has a molecular weight of 141,000 D and92 free amino groups.

[0137] Catalase in a concentration of 2.5 milligrams per milliliter ofbuffer was modified at room temperature. Linear and two-armed mPEGs asdescribed were added at a molar ratio of polymer to protein amino groupsof 5:1 and 10:1, respectively.

[0138] Trypsin in a concentration of 4 milligrams per milliliter ofbuffer was modified at 0° C. Linear and two-armed mPEGs as describedwere added at a molar ratio of polymer to protein amino groups of 2.5:1.

[0139] Asparaginase in a concentration of 6 milligrams per milliliter ofbuffer was modified with linear mPEG at room temperature. Linear mPEG asdescribed was added at a molar ratio of polymer to protein amino groupsof 3:1. Asparaginase in a concentration of 6 milligrams per milliliterof buffer was modified with two-armed mPEG at 37° C. Two-armed mPEG ofthe invention as described was added at a molar ratio of polymer toprotein amino groups of 3.3:1.

[0140] The polymer and enzyme conjugates were purified byultrafiltration and concentrated in an Amicon system with a PM 10membrane (cut off 10,000) to eliminate N-hydroxysuccinimide and reducepolymer concentration. The conjugates were further purified from theexcess of unreacted polymer by gel filtration chromatography on aPharmacia Superose 12 column, operated by an FPLC instrument, using 10mM phosphate buffer of pH 7.2, 0.15 M in NaCl, as eluent.

[0141] Protein concentration for the native forms of ribonuclease,catalase, and trypsin was evaluated spectrophotometrically using molarextinction coefficients of 945×10³ M⁻¹ cm⁻¹, 1.67×10⁵ M⁻¹ cm⁻¹ and3.7×10⁴ M⁻¹ cm⁻¹ at 280 nm, respectively. The concentration of nativeasparaginase was evaluated by biuret assay. Biuret assay was also usedto evaluate concentrations of the protein modified forms.

[0142] The extent of protein modification was evaluated by one of threemethods. The first is a calorimetric method described in Habeeb, A. F.S. A. (1966) Determination of free amino groups in protein bytrinitrobenzensulphonic acid. Anal. Biochem. 14, 328-336. The secondmethod is amino acid analysis after acid hydrolysis. This method wasaccomplished by two procedures: 1) the post-column procedure of Benson,J. V., Gordon, M. J., and Patterson, J. A. (1967) Acceleratedchromatographic analysis of amino acid in physiological fluidscontaining vitamin and asparagine. Anal. Biol. Chem. 18, 288-333, and 2)pre-column derivatization by phenylisothiocyanate (PITC) according toBidlingmeyer, B. A., Cohen, S. A., and Tarvin, T. L. (1984) Rapidanalysis of amino acids using pre-column derivatization. J.Chromatography 336, 93-104.

[0143] The amount of bound linear mPEG was evaluated from norleucinecontent with respect to other protein amino acids. The amount oftwo-armed, mPEG-disubstituted lysine was determined from the increase inlysine content. One additional lysine is present in the hydrolysate foreach bound polymer.

[0144] III. Analysis of Polymer and Enzyme Conjugates

[0145] Five different model enzymes, ribonuclease, catalase,asparaginase, trypsin and uricase, were modified with linear,conventional mPEG of molecular weight 5000 having a norleucine aminoacid spacer arm activated as succinimidl ester and with a two-armed,mPEG-disubstituted lysine of the invention prepared from the samelinear, conventional mPEG as described above in connection with the twostep procedure. The molecular weight of the two-armed mPEG disubstitutedlysine of the invention was approximately 10,000.

[0146] A. Comparison of Enzyme Activity. The catalytic properties of themodified enzymes were determined and compared and the results arepresented in Table 1 below. To facilitate comparison, each enzyme wasmodified with the two polymers to a similar extent by a careful choiceof polymer to enzyme ratios and reaction temperature.

[0147] Ribonuclease with 50% and 55% of the amino groups modified withlinear mPEG and two-armed mPEG, respectively, presented 86% and 94%residual activity with respect to the native enzyme. Catalase wasmodified with linear mPEG and with two-armed mPEG to obtain 43% and 38%modification of protein amino groups, respectively. Enzyme activity wasnot significantly changed after modification. Trypsin modification wasat the level of 50% and 57% of amino groups with linear mPEG and withtwo-armed mPEG, respectively. Esterolytic activity for enzyme modifiedwith linear mPEG and two-armed mPEG, assayed on the small substrateTAME, was increased by the modification to 120% and 125%, respectively.Asparaginase with 53% and 40% modified protein amino groups was obtainedby coupling with linear mPEG and two-armed mPEG, respectively. Enzymaticactivity was increased, relative to the free enzyme, to 110% for thelinear mPEG conjugate and to 133% for the two-armed mPEG conjugate.

[0148] While not wishing to be bound by theory, it is possible that inthe case of trypsin and asparaginase, that modification produces a moreactive form of the enzyme. The K_(m) values of the modified andunmodified forms are similar.

[0149] For the enzyme uricase a particularly dramatic result wasobtained. Modification of uricase with linear mPEG resulted in totalloss of activity. While not wishing to be bound by theory, it isbelieved that the linear mPEG attached to an amino acid such as lysinethat is critical for activity. In direct contrast, modification of 40%of the lysines of uricase with two-armed mPEG gave a conjugate retaining70% activity.

[0150] It is apparent that modification of enzymes with two-armed mPEGgives conjugates of equal or greater activity than those produced byconventional linear mPEG modification with monosubstituted structures,despite the fact that two-armed mPEG modification attaches twice as muchpolymer to the enzyme.

[0151] Coupling two-armed mPEG to asparaginase with chlorotriazineactivation as described in the background of the invention gave majorloss of activity. Presumably the greater activity of enzymes modifiedwith a two-armed mPEG of the invention results because the bulkytwo-armed mPEG structure is less likely than monosubstituted linear mPEGstructures to penetrate into active sites of the proteins. TABLE 1Properties of enzymes modified by linear mPEG and two-armed mPEG. % %NH₂:POLYMER MODIFI- ACTIV- Kcas ENZYME^(a) MOLAR RATIO CATION ITY Km (M)(min⁻¹) Ribonuclease RN  1:0  0 100 RP1   1:2.5 50  86 RP2 1:5 55  94Catalase CN  1:0  0 100 CP1 1:5 43 100 CP2  1:10 38  90 Trypsin^(b) TN 1:0  0 100 8.2 × 830 10⁻⁵ TP1   1:2.5 50 120 7.6 × 1790  10⁻⁵ TP2  1:2.5 57 125 8.0 × 2310  10⁻⁵ Asparaginase AN  1:0  0 100 3.31 × 52310⁻⁶ AP1 1:3 53 110 3.33 × 710 10⁻⁶ AP2   1:3.3 40 133 3.30 × 780 10⁻⁶⁵Uricase UP  1:0  0 100 UP1 1:5 45  0 UP2  1:10 40  70

[0152] Enzymatic activity of native and modified enzyme was evaluated bythe following methods. For ribonuclease, the method was used of Crook,E. M., Mathias, A. P., and Rabin, B. R. (1960) Spectrophotometric assayof bovine pancreatic ribonuclease by the use of cytidine 2′:3′phosphate. Biochem. J. 74, 234-238. Catalase activity was determined bythe method of Beers, R. F. and Sizer, I. W. (1952) A spectrophotometricmethod for measuring the breakdown of hydrogen peroxide by catalase. J.Biol. Chem. 195,133-140. The esterolytic activity of trypsin and itsderivatives was determined by the method of Laskowski, M. (1955)Trypsinogen and trypsin. Methods Enzymol. 2, 26-36. Native and modifiedasparaginase were assayed according to a method reported by Cooney, D.A., Capizzi, R. L. and Handschumacher, R. E. (1970) Evaluation ofL-asparagine metabolism in animals and man. Cancer Res. 30, 929-935. Inthis method, 1.1 ml containing 120 μg of a-ketoglutaric acid, 20 Ul ofglutamic-oxalacetic transaminase, 30 Ul of malate dehydrogenase, 100 μgof NADH, 0.5 μg of asparaginase and 10 μmoles of asparagine wereincubated in 0.122 M Tris buffer, pH 8.35, while the NADH absorbancedecrease at 340 nm was followed.

[0153] B. Proteolytic Digestion of Free Enzyme and Conjugates. The ratesat which proteolytic enzymes digest and destroy proteins was determinedand compared for free enzyme, enzyme modified by attachment of linearactivated mPEG, and enzyme modified by attachment of an activatedtwo-armed mPEG of the invention. The proteolytic activities of theconjugates were assayed according to the method of Zwilling, R., andNeurath, H. (1981) Invertrebate protease. Methods Enzymol. 80, 633-664.Four enzymes were used: ribonuclease, catalase, trypsin, andasparaginase. From each enzyme solution, aliquots were taken at varioustime intervals and enzyme activity was assayed spectrophotometrically.

[0154] Proteolytic digestion was performed in 0.05 M phosphate buffer ofpH 7.0. The free enzyme, linear mPEG and protein conjugate, andtwo-armed mPEG-protein conjugates were exposed to the known proteolyticenzymes trypsin, pronase, elastase or subtilisin under conditions asfollows.

[0155] For native ribonuclease and its linear and two-armed mPEGconjugates, 0.57 mg protein was digested at room temperature with 2.85mg of pronase, or 5.7 mg of elastase, or with 0.57 mg of subtilisin in atotal volume of 1 ml. Ribonuclease with 50% and 55% of the amino groupsmodified with linear mPEG and two-armed mPEG, respectively, was studiedfor stability to proteolytic digestion by pronase (FIG. 1(a)), elastase(FIG. 1(b)) and subtilisin (FIG. 1(c)). Polymer modification greatlyincreases the stability to digestion by all three proteolytic enzymes,but the protection offered by two-armed mPEG is much more effective ascompared to linear mPEG.

[0156] For native and linear and two-armed mPEG-modified catalase, 0.58mg of protein were digested at room temperature with 0.58 mg of trypsinor 3.48 mg of pronase in a total volume of 1 ml. Catalase was modifiedwith linear mPEG and two-armed mPEG to obtain 43% and 38% modificationof protein amino groups, respectively. Proteolytic stability was muchgreater for the two-armed mPEG derivative than for the monosubstitutedmPEG derivative, particularly toward pronase (FIG. 3(a)) and trypsin(FIG. 3(b)), where no digestion took place.

[0157] Autolysis of trypsin and its linear and two-armed mPEGderivatives at 37° C. was evaluated by esterolytic activity of proteinsolutions at 25 mg/ml of TAME. Trypsin modification was at the level of50% and 57% of amino groups with linear mPEG and two-armed mPEG,respectively. Modification with linear mPEG and two-armed mPEG reducedproteolytic activity of trypsin towards casein, a high molecular weightsubstrate: activity relative to the native enzyme was found, after 20minutes incubation, to be 64% for the linear mPEG and protein conjugateand only 35% for the two-armed mPEG conjugate. In agreement with theseresults, the trypsin autolysis rate (i.e., the rate at which trypsindigests trypsin), evaluated by enzyme esterolytic activity, was totallyprevented in two-armed mPEG-trypsin but only reduced in the linearmPEG-trypsin conjugate. To prevent autolysis with linear mPEG,modification of 78% of the available protein amino groups was required.

[0158] For native and linear mPEG- and two-armed mPEG-modifiedasparaginase, 2.5 μg were digested at 37° C. with 0.75 mg of trypsin ina total volume of 1 ml. Asparaginase with 53% and 40% modified proteinamino groups was obtained by coupling with linear mPEG and two-armedmPEG, respectively. Modification with two-armed mPEG had an impressiveinfluence on stability towards proteolytic enzyme. Increased protectionwas achieved at a lower extent of modification with respect to thederivative obtained with the two-armed polymer (FIG. 5).

[0159] These data clearly show that two-armed mPEG coupling is much moreeffective than conventional linear mPEG coupling in providing a proteinwith protection against proteolysis. While not wishing to be bound bytheory, it is believed that the two-armed mPEG, having two polymerchains bound to the same site, presents increased hindrance toapproaching macromolecules in comparison to linear mPEG.

[0160] C. Reduction of Protein Antigenicity. Protein can provoke animmune response when injected into the bloodstream. Reduction of proteinimmunogenicity by modification with linear and two-armed mPEG wasdetermined and compared for the enzyme superoxidedismutase (“SOD”).

[0161] Anti-SOD antibodies were obtained from rabbit and purified byaffinity chromatography. The antigens (SOD, linear mPEG-SOD, andtwo-armed mPEG-SOD) were labelled with tritiated succinimidyl propionateto facilitate tracing. Reaction of antigen and antibody were evaluatedby radioactive counting. In a 500 μL sample, the antigen (in the rangeof 0-3 μg) was incubated with 2.5 μg of antibody. The results show thepractical disappearance of antibody recognition for two-armed mPEG-SOD,while an appreciable antibody-antigen complex was formed for linearmPEG-SOD and native SOD.

[0162] D. Blood Clearance Times. Increased blood circulation half livesare of enormous pharmaceutical importance. The degree to which mPEGconjugation of proteins reduces kidney clearance of proteins from theblood was determined and compared for free protein, protein modified byattachment of conventional, linear activated mPEG, and protein modifiedby attachment of the activated two-armed mPEG of the invention. Twoproteins were used. These experiments were conducted by assaying bloodof mice for the presence of the protein.

[0163] For linear mPEG-uricase and two-armed mPEG-uricase, with 40%modification of lysine groups, the half life for blood clearance was 200and 350 minutes, respectively. For unmodified uricase the result was 50minutes.

[0164] For asparaginase, with 53% modification with mPEG and 40%modification with two armed mPEG, the half lives for blood clearancewere 1300 and 2600 minutes, respectively. For unmodified asparaginasethe result was 27 minutes.

[0165] E. Thermal Stability of Free and Conjugated Enzymes. Thermalstability of native ribonuclease, catalase and asparaginase and theirlinear mPEG and two-armed mPEG conjugates was evaluated in 0.5 Mphosphate buffer pH 7.0 at 1 mg/ml, 9 μg/ml and 0.2 mg/ml respectively.The samples were incubated at the specified temperatures for 15 min., 10min., and 15 min, respectively, cooled to room temperature and assayedspectrophotometrically for activity.

[0166] Increased thermostability was found for the modified forms ofribonuclease, as shown in FIG. 2, at pH 7.0, after 15 min. incubation atdifferent temperatures, but no significant difference between the twopolymers was observed. Data for catalase, not reported here, showed thatmodification did not influence catalase thermostability. A limitedincrease in thermal stability of linear and two-armed mPEG-modifiedasparaginase was also noted, but is not reported.

[0167] F. pH Stability of the Free and Conjugated Enzymes. Unmodifiedand polymer-modified enzymes were incubated for 20 hrs in the followingbuffers: sodium acetate 0.05 M at a pH of from 4.0 to 6.0, sodiumphosphate 0.05 M at pH 7.0 and sodium borate 0.05 M at a pH of from 8.0to 11. The enzyme concentrations were 1 mg/ml, 9 μg/ml, 5 μg/ml forribonuclease, catalase, and asparaginase respectively. The stability toincubation at various pH was evaluated on the basis of enzyme activity.

[0168] As shown in FIG. 2b, a decrease in pH stability at acid andalkline pH values was found for the linear and two-armed mPEG-modifiedribonuclease forms as compared to the native enzyme. As shown in FIG. 4,stability of the linear mPEG and two-armed mPEG conjugates with catalasewas improved for incubation at low pH as compared to native catalase.However, the two-armed mPEG and linear mPEG conjugates showed equivalentpH stability. A limited increase in pH stability at acid and alkaline pHvalues was noted for linear and two-armed mPEG-modified asparaginase ascompared to the native enzyme.

[0169] It should be recognized that there are thousands of proteins andenzymes that can be usefully modified by attachment to the polymerderivatives of the invention. Proteins and enzymes can be derived fromanimal sources, humans, microorganisms, and plants and can be producedby genetic engineering or synthesis. Representatives include: cytokinessuch as various interferons (e.g. interferon-α, interferon-β,interferon-γ), interleukin-2 and interleukin-3), hormones such asinsulin, growth hormone-releasing factor (GRF), calcitonin, calcitoningene related peptide (CGRP), atrial natriuretic peptide (ANP),vasopressin, corticortropin-releasing factor (CRF), vasoactiveintestinal peptide (VIP), secretin, α-melanocyte-stimulating hormone(α-MSH), adrenocorticotropic hormone (ACTH), cholecystokinin (CCK),glucagon, parathyroid hormone (PTH), somatostatin, endothelin, substanceP, dynorphin, oxytocin and growth hormone-releasing peptide, tumornecrosis factor binding protein, growth factors such as growth hormone(GH), insulin-like growth factor (IGF-I, IGF-II), β-nerve growth factor(β-NGF), basic fibroblast growth factor (bFGF), transforming growthfactor, erythropoietin, granulocyte colony-stimulating factor (G-CSF),granulocyte macrophage colony-stimulating factor (GM-CSF),platelet-derived growth factor (PDGF) and epidermal growth factor (EGF),enzymes such as tissue plasminogen activator (t-PA), elastase,superoxide dismutase (SOD), bilirubin oxydase, catalase, uricase andasparaginase, other proteins such as ubiquitin, islet activating protein(IAP), serum thymic factor (STF), peptide-T and trypsin inhibitor, andderivatives thereof. In addition to protein modification, the two-armedpolymer derivative of the invention has a variety of relatedapplications. Small molecules attached to two-armed activated mPEGderivatives of the invention can be expected to show enhanced solubilityin either aqueous or organic solvents. Lipids and liposomes attached tothe derivative of the invention can be expected to show long bloodcirculation lifetimes. Other particles than lipids and surfaces havingthe derivative of the invention attached can be expected to shownonfouling characteristics and to be useful as biomaterials havingincreased blood compatibility and avoidance of protein adsorption.Polymer-ligand conjugates can be prepared that are useful in two phaseaffinity partitioning. The polymers of the invention could be attachedto various forms of drugs to produce prodrugs. Small drugs having themultisubstituted derivative attached can be expected to show alteredsolubility, clearance time, targeting, and other properties.

[0170] The invention claimed herein has been described with respect toparticular exemplified embodiments. However, the foregoing descriptionis not intended to limit the invention to the exemplified embodiments,and the skilled artisan should recognize that variations can be madewithin the scope and spirit of the invention as described in theforegoing specification. The invention includes all alternatives,modifications, and equivalents that may be included within the truespirit and scope of the invention as defined by the appended claims.

What is claimed is:
 1. A polymeric derivative represented by thestructure

wherein poly_(a) and poly_(b) are nonpeptidic and substantiallynonreactive water soluble polymeric arms that may be the same ordifferent, wherein C is carbon, wherein P and Q comprise linkagefragments that may be the same or different and join polymeric armspoly_(a) and poly_(b), respectively, to C by hydrolytically stablelinkages in the absence of aromatic rings in said linkage fragments,wherein R is a moiety selected from the group consisting of H,substantially nonreactive moieties, and linkage fragments havingattached thereto by a hydrolytically stable linkage in the absence ofaromatic rings one or more nonpeptidic and substantially nonreactivewater soluble polymeric arms, and wherein Z comprises a moiety selectedfrom the group consisting of moieties having a single site reactivetoward nucleophilic moieties, sites that can be converted to sitesreactive toward nucleophilic moieties, and the reaction product of anucleophilic moiety and moieties having a single site reactive towardnucleophilic moieties.
 2. The polymeric derivative of claim 1 whereinsaid hydrolytically stable linkages are selected from the groupconsisting of amide, amine, ether, carbamate, thiourea, urea,thiocarbamate, thiocarbonate, thioether, thioester, and dithiocarbamatelinkages.
 3. The polymeric derivative of claim 1 wherein saidnucleophilic moieties are selected from the group consisting of amino,thiol, and hydroxyl moieties.
 4. The polymeric derivative of claim 1wherein said nucleophilic moiety is a biologically active molecule. 5.The polymeric derivative of claim 4 wherein said biologically activemolecule is selected from the group consisting of polypeptides,polynucleotides, and lipids.
 6. The polymeric derivative of claim 1wherein said nucleophilic moiety is a solid surface or a particle. 7.The polymeric derivative of claim 6 wherein said solid particle is aliposome.
 8. The polymeric derivative of claim 1 wherein Z is selectedfrom the group consisting of carboxyl, hydroxyl, activated carboxyl,activated hydroxyl, and conjugates of activated carboxyl or hydroxylsites and molecules having at least one reactive nucleophilic moiety. 9.The polymeric derivative of claim 1 wherein Z comprises a moietyselected from the group consisting of trifluoroethylsulfonate,isocyanate, isothiocyanate, active esters, active carbonates, aldehyde,vinylsulfone, maleimide, iodoacetamide, and iminoesters.
 10. Thepolymeric derivative of claim 9 wherein said active ester isN-hydroxylsuccinimidyl ester and said active carbonates are selectedfrom the group consisting of N-hydroxylsuccinimidyl carbonate,p-nitrophenylcarbonate, and trichlorophenylcarbonate.
 11. The polymericderivative of claim 1 wherein said nonpeptidic polymeric arms areselected from the group consisting of poly(alkylene oxides),poly(oxyethylated polyols), and poly(oxyethylated glucose).
 12. Thepolymeric derivative of claim 1 wherein said nonpeptidic polymeric armsare selected from the group consisting of poly(ethylene glycol),poly(vinyl alcohol), poly(propylene glycol), poly(oxyethylatedglycerol), poly(oxyethylated sorbitol), poly(oxyethylated glucose),poly(oxazoline), poly(acryloylmorpholine), and poly(vinylpyrrolidone).13. The polymeric derivative of claim 1 wherein said nonpeptidicpolymeric arms are linear mPEGs of molecular weight of from about 50 to50,000.
 14. The polymeric derivative of claim 1 wherein said linkagefragments P and Q comprise hydrolytically stable linkages in the absenceof aromatic rings to one or more nonpeptidic and water soluble polymericarms.
 15. The polymeric derivative of claim 1 wherein R comprises alinkage fragment attached by a hydrolytically stable linkage in theabsence of aromatic rings to a nonpeptidic and substantially nonreactivewater soluble polymeric arm.
 16. The polymeric derivative of claim 15wherein R is represented by the general structure —M-poly_(a), whereinpoly_(d) is said polymeric arm and M is said linkage fragment.
 17. Thepolymeric derivative of claim 1 wherein Z further comprises a linkagefragment attached by a hydrolytically stable linkage in the absence ofaromatic rings to a nonpeptidic and substantially nonreactive watersoluble polymeric arm.
 18. A polymeric derivative represented by thestructure

wherein poly_(a) and poly_(b) may be the same or different and areselected from the group consisting of linear poly(ethylene glycol),poly(vinyl alcohol), poly(propylene glycol), poly(oxyethylatedglycerol), poly(oxyethylated sorbitol), poly(oxyethylated glucose),poly(oxazoline), poly(acryloylmorpholine), and poly(vinylpyrrolidone);wherein C is carbon; wherein P and Q comprise linkage fragments that maybe the same or different and join polymeric arms poly_(a) and poly_(b),respectively, to C by hydrolytically stable linkages selected from thegroup consisting of amide, amine, ether, carbamate, thiourea, urea,thiocarbamate, thiocarbonate, thioether, thioester, and dithiocarbamatelinkages; wherein R is a moiety selected from the group consisting of H,substantially nonreactive moieties, and linkage fragments havingattached thereto by a hydrolytically stable linkage in the absence ofaromatic rings one or more nonpeptidic and substantially nonreactivewater soluble polymeric arms; and wherein Z comprises a moiety selectedfrom the group consisting of carboxyl, hydroxyl,trifluoroethylsulfonate, isocyanate, isothiocyanate,N-hydroxylsuccinimidyl ester, N-hydroxylsuccinimidyl carbonate,p-nitrophenylcarbonate, trichlorophenylcarbonate, aldehyde,vinylsulfone, maleimide, iodoacetamide, and iminoesters.
 19. Amulti-armed monofunctional polymeric derivative that is the reactionproduct of at least one monofunctional nonpeptidic polymer derivativeand a linker moiety having two or more active sites that form linkageswith said monofunctional nonpeptidic polymer derivatives in the absenceof aromatic moieties, wherein said linkages between said linker moietyand said monofunctional nonpeptidic polymer derivatives arehydrolytically stable.
 20. The multi-armed monofunctional polymericderivative of claim 19 wherein said linker moiety is selected from thegroup consisting of monohydroxy alcohols and monocarboxylic acids. 21.The multi-armed monofunctional polymer derivative of claim 19 whereinsaid active sites on said linker moiety are nucleophilic moieties. 22.The multi-armed monofunctional polymer derivative of claim 21 whereinsaid nucleophilic moieties are selected from the group consisting ofamino, thiol, and hydroxyl moieties.
 23. The multi-armed monofunctionalpolymer derivative of claim 19 wherein said active sites on said linkermoiety are electrophilic moieties.
 24. The multi-armed monofunctionalpolymer derivative of claim 23 wherein said electrophilic moieties areselected from the group consisting of trifluoroethylsulfonate,isocyanate, isothiocyanate, active esters, active carbonates, aldehyde,vinylsulfone, maleimide, iodoacetamide, and iminoesters.
 25. Themulti-armed monofunctional polymeric derivative of claim 24 wherein saidactive esters are N-hydroxylsuccinimidyl ester and said activecarbonates are selected from the group consisting ofN-hydroxylsuccinimidyl carbonates, p-nitrophenylcarbonates, andtrichlorophenylcarbonates.
 26. The multi-armed monofunctional polymericderivative of claim 19 wherein said hydrolytically stable linkages inthe absence of aromatic rings are selected from the group consisting ofamide, amine, ether, carbamate, thiourea, urea, thiocarbamate,thiocarbonate, thioether, thioester, and dithiocarbamate linkages. 27.The multi-armed monofunctional polymeric derivative of claim 19 whereinsaid monofunctionality is selected from the group consisting ofcarboxyl, hydroxyl, activated carboxyl, activated hydroxyl, andconjugates of activated carboxyl or hydroxyl sites and molecules havingat least one reactive nucleophilic moiety.
 28. The multi-armedmonofunctional polymeric derivative of claim 19 wherein saidmonofunctionality is selected from the group consisting oftrifluoroethylsulfonate, isocyanate, isothiocyanate, active esters,active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, andiminoesters.
 29. The multi-armed monofunctional polymeric derivative ofclaim 28 wherein said active ester is N-hydroxylsuccinimide and saidactive carbonates are selected from the group consisting ofN-hydroxylsuccinimide carbonates, p-nitrophenylcarbonates, andtrichlorophenylcarbonates.
 30. The multi-armed monofunctional polymericderivative of claim 19 wherein said nonpeptidic polymeric derivative isselected from the group consisting of poly(alkylene oxides),poly(oxyethylated polyols), and poly(oxyethylated glucose).
 31. Themulti-armed monofunctional polymeric derivative of claim 19 wherein saidnonpeptidic polymer derivative is selected from the group consisting ofactivated poly(ethylene glycol), poly(vinyl alcohol), poly(propyleneglycol), poly(oxyethylated glycerol), poly(oxyethylated sorbitol),poly(oxyethylated glucose), poly(oxazoline), poly(acryloylmorpholine),and poly(vinylpyrrolidone).
 32. The multi-armed monofunctional polymericderivative of claim 19 wherein said nonpeptidic polymer derivative is alinear mPEG of molecular weight of from about 50 to 50,000 and themulti-armed monofunctional polymeric derivative has two arms of saidlinear mPEG.
 33. A material comprising a solid surface or particlehaving attached thereto compounds of the structure claimed in claim 19 .34. The material of claim 33 wherein said solid surface or particle is aliposome.
 35. A biologically active structure comprising a biologicallyactive molecule having attached thereto one or more compounds of thestructure claimed in claim 19 .
 36. The biologically active structure ofclaim 35 wherein said biologically active molecule is selected from thegroup consisting of polypeptides, polynucleotides, and lipids.
 37. Thebiologically active structure of claim 36 wherein said polypeptide isselected from the group consisting of asparaginase, catalase,ribonuclease, subtilisine, trypsin, and uricase.
 38. A two-armedpolymeric derivative having a structure selected from the groupconsisting of:

wherein poly_(a) and poly_(b) may be the same or different and comprisemoieties selected from the group consisting of poly(ethylene glycol),poly(vinyl alcohol), poly(propylene glycol), poly(oxyethylatedglycerol), poly(oxyethylated sorbitol), poly(oxyethylated glucose),poly(oxazoline), poly(acryloylmorpholine), and poly(vinylpyrrolidone)moieties; and wherein Z comprises a moiety selected from the groupconsisting of moieties having a single site reactive toward nucleophilicmoieties, sites that can be converted to sites reactive towardnucleophilic moieties, and the reaction product of a nucleophilic moietyand moieties having a single site reactive toward nucleophilic moieties.39. The two-armed polymeric derivative of claim 38 wherein said reactivesite is selected from the group consisting of carboxyl, activatedcarboxyl, hydroxyl, activated hydroxyl, and conjugates of activatedcarboxyl or hydroxyl sites and molecules having at least one reactivenucleophilic moiety.
 40. The polymeric derivative of claim 38 wherein Zcomprises a moiety selected from the group consisting oftrifluoroethylsulfonate, isocyanate, isothiocyanate, active esters,active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, andiminoesters.
 41. The polymeric derivative of claim 40 wherein saidactive ester is N-hydroxylsuccinimidyl ester and said active carbonatesare selected from the group consisting of N-hydroxylsuccinimidylcarbonate, p-nitrophenylcarbonate, and trichlorophenylcarbonate.
 42. Amolecule having the structure

wherein mPEG_(a) and mPEG_(b) have the structureCH₃—(CH₂CH₂O)_(n)CH₂CH₂—, wherein n equals from 1 to about 1,150, andwherein n may be the same or different for mPEG_(a) and mPEG_(b). 43.The molecule of claim 42 wherein n equals from 1 to about
 570. 44. Amethod of synthesizing a multi-armed, water soluble, monofunctionalpolymeric molecule comprising reacting one or more nonpeptidicmonofunctional polymers of the structure poly-W, wherein W is an activemoiety providing the monofunctionality for the polymer, with a linkermoiety having two or more active sites with which W is reactive, andforming hydrolytically stable linkages in the absence of aromatic ringsbetween the monofunctional polymer and the linker moiety at the linkermoiety active sites, the linker moiety having a reactive site for whichsaid active moiety —W is not reactive to provide the monofunctionalityfor the multi-armed molecule.
 45. The method of claim 44 wherein themethod further comprises activating the reactive site after themulti-armed polymeric compound is formed with an electrophilic moiety.46. The method of claim 45 wherein the electrophilic moiety is reactivewith nucleophilic moieties selected from the group consisting of amino,thiol, and hydroxyl moieties.
 47. The method of claim 44 wherein theactive moiety W is an electrophilic moiety selected from the groupconsisting of trifluoroethylsulfonate, isocyanate, isothiocyanate,active esters, active carbonates, aldehyde, vinylsulfone, maleimide,iodoacetamide, and iminoesters.
 48. The method of claim 47 wherein theactive ester is N-hydroxylsuccinimidyl ester and the active carbonatesare selected from the group consisting of N-hydroxylsuccinimidylcarbonate, p-nitrophenylcarbonate, and trichlorophenylcarbonate.
 49. Themethod of claim 44 wherein the active moiety W is a nucleophilic moietyselected from the group consisting of amino, thiol, and hydroxylmoieties.
 50. The method of claim 44 wherein the active sites on thelinker moiety are nucleophilic moieties selected from the groupconsisting of amino, thiol, and hydroxyl moieties.
 51. The method ofclaim 44 wherein the active sites on the linker moiety are electrophilicmoieties selected from the group consisting of trifluoroethylsulfonate,isocyanate, isothiocyanate, active esters, active carbonates, aldehyde,vinylsulfone, maleimide, iodoacetamide, and iminoesters.
 52. The methodof claim 51 wherein the active ester is N-hydroxylsuccinimidyl ester andthe active carbonates are selected from the group consisting ofN-hydroxylsuccinimidyl carbonate, p-nitrophenylcarbonate, andtrichlorophenylcarbonate.
 53. The method of claim 44 wherein thehydrolytically stable linkages are selected from the group consisting ofamide, amine, ether, carbamate, thiourea, urea, thiocarbamate,thiocarbonate, thioether, thioester, and dithiocarbamate linkages.
 54. Amethod for preparing a polymeric derivative represented by the structure

comprising the steps of: a) reacting nonpeptidic, water soluble,monofunctional polymers of the structure poly_(a)-W and poly_(b)-W witha linker moiety having at least two active sites for which W isselective, a reactive site Z for which W is not selective, and a moietyR which is substantially nonreactive, wherein W is an activeelectrophilic moiety selected from the group consisting oftrifluoroethylsulfonate, isocyanate, isothiocyanate, active esters,active carbonates, aldehyde, vinylsulfone, maleimide, iodoacetamide, andiminoesters, and may be the same or different on poly_(a) and poly_(b),wherein poly_(a) and poly_(b) are polymer moieties selected from thegroup consisting of poly(ethylene glycol), poly(vinyl alcohol),poly(propylene glycol), poly(oxyethylated glycerol), poly(oxyethylatedsorbitol), poly(oxyethylated glucose), poly(oxazoline),poly(acryloylmorpholine), and poly(vinylpyrrolidone) and may be the sameor different, and wherein the active sites of the linker moiety arenucleophilic sites selected from the group consisting of amino, thiol,and hydroxyl; and b) forming hydrolytically stable linkages P and Q,which may be the same or different, in the absence of aromatic ringsbetween the polymer and the linker moiety that are selected from thegroup consisting of amide, amine, ether, carbamate, thiourea, urea,thiocarbamate, thiccarbonate, thioether, thioester, and dithiocarbamatelinkages.
 55. The method of claim 54 wherein the linker moiety issubstituted with polymer at each active site in one step.
 56. The methodof claim 55 wherein the linker moiety is substituted with polymer ateach active site in more than one step.
 57. The multi-armed polymericderivative of claim 54 wherein said linker moiety is selected from thegroup consisting of monohydroxy alcohols and monocarboxilic acids havingtwo or more active moieties selected from the group consisting of thiol,amino, and hydroxyl moieties.
 58. The multi-armed polymeric derivativeof claim 1 wherein Z is selected from the group consisting of carboxyl,hydroxyl, activated carboxyl, activated hydroxyl, and conjugates ofprecursor activated carboxyl or hydroxyl sites and molecules havingsites for which said precursor activated sites are active.
 59. A methodfor forming monofunctional monomethoxy-poly(ethylene glycol)disubstituted lysene comprising the following step:


60. The method of claim 59 wherein the reaction takes place in water ata pH of about 8.0.
 61. The method of claim 59 further comprising thesteps of


62. The method of claim 61 wherein steps a) and b) take place inmethylene chloride.
 63. The method of claim 59 further comprising thesteps of activating the carboxyl moiety and reacting the activatedcarboxyl moiety with an active moiety to join the disubstituted lysineto the active moiety.
 64. A method for forming a monofunctionalmonomethoxy-poly(ethylene glycol) disubstituted compound comprising thefollowing steps:


65. The method of claim 64 further comprising the steps of activatingthe carboxyl moiety and reacting the activated carboxyl moiety with anactive moiety to join the disubstituted lysine to the active moiety. 66.The method of claim 64 wherein step a) takes place in aqueous buffer.67. The method of claim 64 wherein step b) takes place in methylenechloride.